Actinobacillus actinomycetemcomitans (A.a.) is a nonenteric gram negative bacterium which is associated with a variety of infectious disease processes including endocarditis, subcutaneous abscesses, and several forms of periodontal disease (Block et al., 1973, A, J, Med. Sci. 276:387-392; Page et al., 1966, N. Engl. J. Med. 275:181-188; Zambon, 1985, J. Clin. Periodontal. 12:1-20; Zambon et al., 1983, Infect. Immun. 41:19-27). Juvenile periodontitis is an oral disease which afflicts adolescents and pre-teens and is caused by A.a. Symptoms include inflammation of the gingiva and destruction of the tissues supporting the teeth.
A.a. produces a variety of potential virulence factors including a proteinaceous leukotoxin (Kraig et al., 1990, Infect. Immun. 58:920-929; Lally et al., 1991, Microb. Pathog. 111-121) which is a member of the RTX (repeats-in-toxin) family of bacterial cytolysins (Welch, 1991, Mol. Microbiol.5:521-528). A.a. leukotoxin exhibits a unique cytolytic specificity in that it destroys human polymorphonuclear leukocytes and macrophages, but does not lyse other types of cells, for example, epithelial and endothelial cells, fibroblasts, erythrocytes and platelets (Baehni et al., 1981, Arch. Oral Biol. 27:671-676; Taichman et al., 1980, Infect. Immun. 28:258-278; Taichman et al., 1984, J. Period. Res. 19:133-145; Taichman et al., 1987, Oral Microbiol. Immun. 2:97-104).
The A.a. leukotoxin is expressed from an operon comprising four genes designated 1txC, 1txA, 1txB, and 1txD. The structural gene encoding the A.a leukotoxin (1txA), encodes a 116 kDa peptide which is approximately 40 and 50% identical to the Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin (Kraig et al., 1990, Infect. Immun. 58:920-929; Lally et al., 1989, Biochem. Biophys. Res. Commun. 159:256-272). The structure of the actual A.a. leukotoxin is similar to other RTX toxins based upon DNA sequence homology and on similarities in the mechanism of action of this class of toxins (Felmlee et al., 1985, J. Bacteriol. 163:94-105; Felmlee et al., 1988, Proc. Natl. Acad. Sci. USA 85:5279-5273; Ludwig et al., 1988, Mol. Gen. Genet. 214:553-561; Ludwig et al., 1987, Mol. Gen. Genet. 206:238-245). However, the A.a. leukotoxin differs from other RTX toxins in that it is not secreted from the bacterial cell (Lally et al., 1991, Microb. Pathog. 11:111-121). The products of the three remaining 1x genes, 1txB, 1txC and 1txD, are believed to be required for activation and transport of the leukotoxin in the cell.
At least two mRNAs are transcribed from the 1txCABD gene cluster (Spitznagel et al., 1991, Infect. Immun. 59:1394-1401). A predominant mRNA of 3.8 kb encodes 1txC and 1txA, and a less abundant mRNA of 8 kb encoding 1txCABD results when transcription fails to terminate at a rho-independent transcriptional terminator situated between 1txA and 1txB (Lally et al., Microb. Pathog. 11:111-121; Spitznagel et al., 1991, Infect. Immun. 59:1394-1401). Expression of leukotoxin varies among different strains of A.a. Analysis of several nonleukotoxic strains has revealed that they possess the 1tx operon but express only low steady state levels of 1tx mRNA relative to the highly leukotoxic strain, JP2 (Spitznagel et al., 1991, Infect. Immun. 59:1394-1401). The basis for this difference was unknown prior to the instant invention.
Thus, it is known that two populations of A.a. exist in the oral cavity, one producing high levels of leukotoxin and another producing low levels of leukotoxin. An understanding of the molecular basis for the difference between these two populations has provided a means for which there has been a long felt need, of differentiating between the two populations in order that appropriate treatment may be administered to a patient infected with a leukotoxic strain of A.a., which patient may or may not have progressed to severe periodontal disease.